We have been developing tools and resources that make it possible to analyze a large number of genes in various experimental conditions. In our earlier work, we 1) constructed cDNA libraries from early mouse embryos and stem cells and generated a large number of expressed sequence tags (ESTs), 2) developed a glass-slide microarray platform containing in situ-synthesized 60-mer oligonucleotide probes representing approximately 44,000 unique mouse transcripts, 3) produced the web-based ANOVA-FDR software to provide user-friendly microarray data analysis, and 4) developed an algorithm and a fully-automated computational pipeline for transcript assembly from expressed sequences aligned to the mouse genome. In addition, we recently developed a comprehensive database and web browser of the binding sites of transcription factors (TFs) and cis-regulatory modules (CRMs) on the mouse genome. These resources and tools are now applied to the systematic analysis of gene regulatory networks in mouse embryonic stem cells. In our pilot project, we have demonstrated that it is possible to analyze and identify downstream target genes by monitoring the global gene expression patterns of mouse ES cell lines, when a gene encoding a specific TF (Pou5f1 or Oct4 in this case) is manipulated so that the gene can be overexpressed or repressed. We have extended this strategy to other key transcription factors (Sox2 and Nanog) in mouse ES cells. To further facilitate the analysis of gene regulatory networks, we have recently carried out the systematic repression of 105 TFs by transient transfection gene-specific shRNAs in mouse ES cells. We have then carried out microarray-based gene expression profiling 72 hours after transfection. We are currently analyzing these microarray data.